Inflammation is an essential protective mechanism that must be tightly regulated to ensure that responses are of a suitable magnitude and duration. Dysregulated inflammation underlies chronic diseases (e.g. inflammatory bowel disease). By generating or degrading inflammatory mediators and by cleaving receptors and channels, proteases both initiate and terminate inflammation. This proposal examines the importance of extracellular and intracellular proteolysis in the regulation of inflammatory signaling that underlies inflammatory bowel disease. Aim 1 examines the hypothesis that proteases which are activated in colitis play a major causative role in inflammation. Since proteases are regulated by control of activity, rather than by expression, approaches are required that profile protease activity in disease. Activity-based probes, which interact only with active proteases, are a major conceptual and technological advance in identifying protease networks in disease. By using biotin-tagged probes and proteomics, and near infrared-tagged probes and whole body and cellular imaging, activated proteases will be identified and localized in mice with colitis. Inhibitors will be used to define the causative role of proteases in colitis. Aim 2 examines the hypothesis that extracellular proteases signal to colonocytes by cleaving basolateral protease-activated receptors (PARs) and apical epithelial sodium channel (ENaC), mediators of inflammatory signaling and electrolyte transport. Studies at the molecular and cellular level will examine the innovative concepts that protease-induced inflammatory signals depend on the site of proteolysis within an individual PAR and the spectrum of PARs that are activated by an individual protease. The role of PARs in colitis will be investigated using novel blocking antibodies, which circumvents the lack of small molecular antagonists. Electrophysiological approaches will determine whether proteases that are activated in the lumen of the inflamed colon cleave apical ENaC and promote sodium absorption, which protects against electrolyte and fluid loss. Aim 3 examines the hypothesis that endosomes are platforms for sustained PAR inflammatory signaling and that endosomal proteases play a critical role in regulating the duration and magnitude of this signaling. By studying cultured colonocytes by subcellular fractionation and proximity assays of protein-protein interactions, studies will examine the innovative concept that PARs and 2- arrestins assemble signaling complexes in endosomes that transmit sustained inflammatory signals. The importance of this mechanism in colitis will be examined in 2-arrestin null mice. Since endosomal signals must be tightly regulated to avoid uncontrolled inflammation, experiments will determine whether endosomal proteases that cleave PARs or deubiquitinate 2-arrestins terminate endosomal signaling. The proposal will thus identify new mechanisms of protease-regulated inflammation, with implications for non-invasive detection of inflammation prior to irreversible damage, and to improved and more specific therapy. The results will provide insight into inflammatory bowel disease that are applicable to inflammatory diseases that affect all systems. PUBLIC HEALTH RELEVANCE: Chronic inflammation underlies diseases that affect all organ systems that are a major cause of morbidity and mortality. Difficulties in effective treatment are related to inadequate approaches for the early detection of disease prior to irreversible organ damage, and an incomplete understanding of the mechanisms that control inflammatory signaling. This proposal seeks to determine the role of proteases in chronic inflammation of the intestine. Studies will pioneer new methods for the early detection of inflammation by non-invasive approaches prior to irreversible organ damage, and will define the causative role of proteases in signaling inflammation. The proposal will provide new information which is beneficial for the detection, diagnosis and treatment of inflammatory diseases that are a global cause of morbidity and mortality.